Paper strip test for citrate utilization

ABSTRACT

A PAPER STRIP MATERIAL IS IMPREGNATED IN CONSECUTIVE ZONES WITH (1) A REAGENT MEDIUM CONSISTING OF A CITRATE SOURCE SUCH AS CITRIC ACID OR A CITRATE SALT, AND A BUFFER SYSTEM WHICH MAINTAINS THE PH OF THIS REAGENT MEDIUM AT ABOUT 6.4; (2) A MICROBIOLOGICALLY INERT HYDROPHOBIC BARRIER; (3) A PH INDICATOR OR A COMBINATION OF INDICATORS WHICH CHANGES COLOR BETWEEN A PH OF 6 AND PH OF 8. SUITABLE BUFFERS INCLUDE A PHOSPHATE BUFFER PREPARED FROM MONOBASIC POTASSIUM PHOSPHATE AND DIBASIC POTASSIUM PHOSPHATE; A GOMORI TRIS-MALEATE BUFFER, FORMED FROM TRIS-(HYDROXY METHYL) AMINOMETHANE AND MALEIC AICD; AND A KOLTHOFF BORAX-PHOSPHATE BUFFER WHICH IS A COMBINATIN OF SODIUM BORATE AND MONOBASIC POTASSIUM PHOSPHATE. SUITABLE INDICATORS INCLUDE: A COMBINATION OF BROM THYMOL BLUE AND A WATER SOLUBLE GRADE OF PHENOL RED; NEUYTRAL RED; PHENOL RED; ROSLIC ACID; M-DINITROBENZOYLENE UREA; BRILLIANT YELLOW; AND QUINOLINE BLUE. THE REAGENT MEDIUM AND THE INDICATOR SYSTEM ARE ADJUSTED TO A PH OF 6.4 BEFORE APPLICATION TO TH EPAPTER STRIP. CULTRUES GROWN ON A NON-CITRATE CONTAINING MEDIUM CAN BE TESTED FOR CITRATE UTILIZATION BY INCUBATIN IN CONTACT WITH THE REAGENT ZONE OF THE PAPER STGRIP FOR APPROXIMATELY 4 HOURS, AND THEN CONTACTING THE CULTURE WITH THE INDICATOR ZONE OF THE PAPER STRIP AND OBSERVING THE COLOR CHANGE APPROXIMATELY ONE-HALF HOUR LATER.

United States Patent U.S. Cl. 195103.5 6 Claims ABSTRACT OF THE DISCLOSURE A paper strip material is impregnated in consecutive zones with (l) a reagent medium consisting of a citrate source such as citric acid or a citrate salt, and a butter system which maintains the pH of this reagent medium at about 6.4; (2) a microbiologically inert hydrophobic barrier; (3) a pH indicator or a combination of indicators which changes color between a pH of 6 and a pH of 8. Suitable buffers include a phosphate buffer prepared from monobasic potassium phosphate and dibasic potassium phosphate; a Gomori tris-maleate butler, formed from tris-(hydroxy methyl)aminomethane and maleic acid; and a Kolthoff borax-phosphate buffer which is a combination of sodium borate and monobasic potassium phosphate. Suitable indicators include: a combination of Brom Thymol Blue and a water soluble grade of Phenol Red; Neutral Red; Phenol Red; Rosolic Acid; m-Dinitrobenzoylene Urea; Brilliant Yellow; and Quinoline Blue. The reagent medium and the indicator system are adjusted to a pH of 6.4 before application to the paper strip. Cultures grown on a non-citrate containing medium can be tested for citrate utilization by incubation in contact with the reagent zone of the paper strip for approximately 4 hours, and then contacting the culture with the indicator zone of the paper strip and observing the color change approximately one-half hour later.

CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of our copending application, U.S. Ser. No. 632,894, filed Apr. 24, 1967, now abandoned.

BACKGROUND OF THE INVENTION The citrate utilization test is based on the fact that certain micro-organisms are capable of utilizing sodium citrate as their sole source of carbon. They will grow in a chemically defined medium in which sodium citrate is the only carbon compound present. Certain unknown alkaline metabolites are produced during growth and a measurable pH change in the culture medium results. Other micro-organisms do not utilize citrate in this manner and no pH change occurs.

The citrate utilization test is well known in the field of microbiological testing. It is one of a series of tests known collectively as IMViC reactions (I=indol, M=methyl red, V=Voges-Proskauer reaction and C=citrate). These tests are used, for the most part, to ditferentiate the various organisms in the family Enterobacteriaceae. The ditferentiation of Salmonella from Shigella is particularly important in diagnostic bacteriology; and the diiferentiation of Aerobacter-Klebsiella from Escherichia coli is of significance in testing water for potability and in diagnostic bacteriology. The presence of E. coli in water indicates fecal contamination. In the conventional Simmons citrate test, one indicator is used which changes color with pH change as a positive reaction to citrate utilization (J. Infectious Diseases 39: 209-214, 1926). With the Simmons test, the incubation time may have to be extended for four days to insure detection of delayed positive reactions. This is obviously highly undesirable in a clinical laboratory.

Hargove and Weaver modified the Simmons test, varying the composition of the growth medium for the development of a rapid microtechnic (Am. J. Clin. Path. 21: 286289, 1951). Hargrove and Weaver suggest that cultures for inoculation should be grown on a culture medium containing citrate as one of the sources of carbon, to limit the incubation time required to 10' to 12 hours. However, growing cultures on a citrate containing medium prior to incubation for the citrate test can require as much as forty-eight hours, hence, actual time save is negligible. Tests made with cultures grown on a non-citrate containing medium require an incubation time of 20 to 48 hours. The indicator Brom Thymol Blue, used in the above Hargrove and Weaver modification, is not incorporated in the culture medium but added after incubation, since it tends to be reduced by bacterial action.

After the addition of Brom Thymol Blue a reaction period of minutes is adequate for a positive result, indicated by a deep blue coloration. A definite green color is considered a negative reaction. At times, an indeterminate bluish-green color develops. For some positive cultures, an additional reaction time of two hours will resolve the problem, since some organisms utilize the citrate in this reaction at a slower rate. However, the detection of certain species remains diflicult.

It is obvious that there are many disadvantages connected with the Hargrove-Weaver modification of the citrate test. lengthy test procedures involving storage and repeated handling of test samples tend to minimize the effectiveness of any bacteriological laboratory testing. For instance, when the community water supply is under study, considerable illness may result due to lengthy test procedures. Any delay in determination of the identity of microorganisms can be a health hazard, since it may be some time before corrective measures, if necessary, are initiated.

In an attempt to develop a rapid paper strip test for citrate utilization, known citrate test systems were investigated by application to bibulous materials. None of these products were found to be a significant improvement over prior methods.

SUMMARY OF THE INVENTION This invention relates to an impregnated paper strip for the rapid and positive indenti-fication of citrate utilization by micro-organisms. Consecutive zones in the paper strip are impregnated with: (1) a reagent medium consisting of a citrate source such as citric acid or a citrate salt, and a buffer system, which maintains the pH of this reagent medium at about 6.4, selected from the group: a phosphate bulfer prepared from monobasic potassium phosphate and dibasic potassium phosphate; a Gomori tris-maleate buffer, formed from tris-(hydroxy methyDaminomethane and maleic acid; and a Kolthoif borax-phosphate buifer which is a combination of sodium borate and monobasic potassium phosphate; (2) a microbiologically inert hydrophobic barrier; and (3) a pH indicator or a combination of indicators which changes color between a pH of about 6 and a pH of about 8, selected from the group: Brom Thy mol Blue and a water soluble grade of Phenol Red; Neutral Red; Phenol Red; Rosolic Acid; m-Dinitrobenzoyl-ene Urea; Brilliant Yellow; and Quinoline Blue.

The reagent medium and the indicator system are adjusted to a pH of about 6.4 before application to the paper strip by the addition of an acid or base which does not interfere with the microbiological reaction.

DESCRIPTION OF THE INVENTION It is the primary object of this invention to provide a diagnostic preparation which permits the rapid and positive identification of micro-organisms utilizing citrate in culture media.

It is another object of this invention to provide an improved citrate utilization test wherein results are obtained in approximately 4.5 hours, in spite of the fact that the cultures for inoculation are not grown in a citrate-containing culture medium.

It is yet another object of this invention to provide a more definite color response to indicate positive and negative reactions to citrate utilization.

Other objects and advantages of this invention will become apparent from the following detailed description.

We have now found that cultures grown on a noncitrate containing medium can be tested for utilization of citrate by incubating a large inoculum of the microorganisms at a temperature ranging from about 34 to 37 C. for a period of time of from about three to six hours in the presence of the citrate containing reagent zone of the diagnostic test strip of this invention. Actually the incubation time and temperature can be varied widely to achieve growth of organisms and subsequent production of citrate. However, incubating for more than six hours, which would be required for temperatures lower than 34 C., reduces the time advantage over the standard test procedures obtained by incubating at higher temperatures. Further, while incubating at temperatures above 37 C. is possible with some organisms, this in undesirable, since one is identifying unknown organisms some of which cannot live at higher than 39 C. Fluctuations in typical incubation baths are usually :2 C., so for reliable results, about 37 C. has been selected as the safest high temperature.

For the preferred embodiment of our invention, a large inoculum of the unknown microorganisms is incubated at about 37 C. for at least four hours in the presence of the citrate-containing reagent and then brought into contact with the indicator or combination of indicators for at least thirty minutes.

We have also found that an advantageous carrier for both the reagent medium and the indicator system is a strip of bibulous material impregnated over a portion of its area with the reagent medium and over another portion of its area with the indicator combination system. These impregnated zones are separated by a hydrophobic barrier zone.

While any suitable source of citrate which can be utilized by the microbiological organisms is applicable in this invention, we have found that citric acid, sodium citrate and potassium citrate are preferred for the reagent medium. Actually, the use of citric acid requires the subsequent addition of an alkaline material which is micro= 4 biologically inert in order to bring the pH of the reaction medium to about 6.4 for optimum utilization of the citrate by the micro-organisms under study. Suitable microbiologically inert, alkaline materials include sodium, potassium, and ammonium hydroxide. These bases, when added to a citric acid solution, form the citrate salt. Therefore, the initial use of a citrate salt such as sodium or potassium citrate is the preferred source of citrate for the reagent medium. Approximately 8 to 12 grams of citrate dissolved in distilled water to yield about milliliters of solution has been found to provide suflicient citrate when applied to a strip of bibulous material for utilization by micro-organisms under study.

A buffer is included in the reagent medium to maintain the optimum pH of about 6.4. The production of alkaline metabolites by micro-organisms utilizing the citrate will, of course, raise the pH of the system. This is the measurable change which indicates a positive reaction to the test. The presence of the buffer, however, prevents identification of false positives which can result from such factors as cell lysis. Buffers which do not adversely affect the growth of the micro-organisms under study and which give a pH of about 6.4 are required. The following are suitable: a phosphate buffer prepared from mono-basic potassium phosphate and dibasic potassium phosphate; a Gomori tri-maleate buffer, formed from tris-(hydroxy methyl)aminomethane andmaleic acid; and a Kolthoff borax-phosphate buffer which is a combination of sodium borate and monobasic potassium phosphate. The makeup of the buffer must be such that the system to which it is added resists alteration of a pH of about 6.4. The following buffer ingredients, when added to fromabout 8 to about 12 grams of the citrate source in a distilled water solution of about 100 milliliters, have been found to give an effective reagent medium which can be applied successfully to our bibulous test strips:

Each system is, of course, adjusted to a pH of about 6.4 by the addition of a sufficient amount of an acid or base which does not interfere with the microbiological reaction, e.g., hydrochloric, nitric, or phosphoric acid and sodium, potassium, or ammonium hydroxide, respectively.

The preferred reagent medium for application to our bibulous test strip contains, in each distilled water solu tion of about 100 milliliters, the following ingredients:

(a) from about 8 to 12 grams sodium citrate,.

,(b) about 2.5 grams monobasic potassium phosphate,

and

(c) about 0.5 gram dibasic potassium phosphate.

The pH of the above reagent medium is adjusted-to about 6.4 by the addition of a sufiicient amount of one of the above mentioned acids or bases which does not interfere with the microbiological reaction The most preferred reagent medium contains in each distilled water solution of about 100milliliters:

(a) about 10 grams sodium citrate, i v Y (b) about 2.5 grams monobasic potassium phosphate, (0) about 0.5 gram dibasic potassium phosphate, and (d) sufiicient 1 N hydrochloric acid, ,or 1 N sodium hydroxide to adjust the pH to about 6.4.

A pH indicator or combination of pH indicators which provides a reliable and distinct color change between a pH of about 6'to about 8, in the presence of the growing microorganisms, is applied to our diagnostic test strip to indicate positive or negative citrate utilization. The fol- Of the above, the most preferred indicator solution is lowing H indicators perform this function and provide Number 1, the Brom Thymol Blue and Phenol Red com- P a stable test product when applied to our bibulous test bination. strip. Stability is sufficient to allow marketability and In the preferred embodiment of our invention, using insure reliability of the marketed product. the Brom Thymol Blue/ Phenol Red combination, an terim pH adjustment is made during the preparation of (1) Brom Tymol Blueand Phenol Red, water soluble the indicator system. A solution of the Brom Thymol Z ZQE brown to p mp 1e betwwn a PH of about Blue in the ethyl alcohol, to which about 25 m1. of the (2) N distilled water has been added, is brought to a measured I pH of about 6.4 (as determined on a Beckman pH meter) 2 i g red to yellow between a PH of about by the addition of 0.1 N sodium hydroxide. After adding (3) P'henol k the Phenol Red and 'sufiicient distilled water to bring the volume to about 100 ml., it is necessary to adjust the pH g ii g 2 willow to red between a PH of about once again to about 6.4 by the addition of 0.1 N hydro- (4) ,R'osolic acid chloric acid. It has been found that this step-by-step procedure materially improves the workability of the product. 2 5 15 g brown to red between a pH of about The hydrophobic barrier composition must prevent the leaching of the culture medium or reagent across the bibulous material into the indicator zone during incubaa tion. It should also be microbiologically inert in this sys- ;E g tem. Any substance which will form a water-proof barrier a of this type may be used. Suitable materials include changes from yellow to orange between a PH of waxes, lacquers, and plastics, particularly a colorless abQut to polymerized methyl methacrylate coating composition (7) Qumohne Blue sold under the trade name Krylon 150 Crystal Clear by Krylon, -Inc., Norri'stown, Pa. The Krylon material is particularly preferred. It is supplied in a toluene vehi- (5) mDinit'robenzoylene Urea changes from colorless to yellow between a pH of changes from colorless to blue between a pH of about 6.6 to 8.6

Solutions of the indicators are made up for application cle and may be diluted for ease of application with addito the bibulous material. Distilled water is the usual soltional toluene or other hydro-carbon thinners, such as vent, but for certain indicators, an additional solvent, such ethyl, methyl, or propyl alcohol U.S.P. as methyl, ethyl, propyl or butyl alcohol may be needed It has been found that about 100 ml. of barrier solution to aid in obtaining a solution. The compositions of indimade from 75 to 100 ml. Krylon and 0 to 25 ml. diluent cator solutions are listed in the two table below; is suitable. A part1cularly preferred combination is pre- TABLE 1 Broad range Indicator composition (grams) Solvent J Brom Thymol Blue About 0.l8-0.22 .}22.5 to 27.5 ml. of 1 4C alcohol, q.s. to about 1 """lPhenol Red, Water-soluble About 0.18-0.22. 100 ml. with distilled Water.

Neutral Red.. About 0.2-0.7 5-60 ml. of l-4C alcohol, q.s. to about 100 ml. with distilled Water. 3 Phenol Red About 0025-0075.-- 30-90 ml. of 1-40 alcohol, q.s. to about 100 ml. with dfistillgd watleri t b 00 About 0.20.6 90-100 ml. 0 14 a co 0 q.s. o a out 1 Rosohc 801d ml. with distilled Water. 5; m-Dinitrobenzoyleno urea About 0. 0.7 Q.s. to about 100 ml. with distilled Water. 6. Brilliant Yellow About 0.751.5 Do. 7. Quinoline Blue About 0.2-1.2". Q.s. to about 100 ml. with 100% ethanol.

The-indicator solution is adjusted to a measured pH pared from about 85 KTYIQII With about 15 ethyl of about 6.4, by the addition of a sufficient amount of alcohol the l b 'm acids or bases and applied to The blblllOllS materials suitable for the practice of this theindicator .zone of .our test strip. When brought into mvPntlon are those mateFIaIF, Whlch, y f flns of capillary fi with the microbiological culture under Study a action, are able to hold liquid. such materials include filter distinct-color can be observed for a positive reaction as paper felt porous ceramlc stnpst Woven or matted glass fib dthl'k.A ct 11 d the culture medium reaches an alkaline pH between about iggg 2 5 g: 15 3 2; 5335 5? er ls Eaton pH 7.4 to about 7.6 due to the production of alkaline It is possible to prepare a diagnostic product from a metabolites aS th6 C1tIat is utilized by the growing microbibulous material having only three zones: a reagent zone,

organism: the citrate is not utilized an acid color of a barrier zone and an indicator zone, wherein the third the indicator is maintained to indicate anegative reaction. Zone containing th i di to i t th d f th t t p e e e l g for the gredients of the indicator strip. Handling of-such a three-zone test strip is awkward solutions of'this invention are: since there is some danger of alteration of the pH of the TABLE 2 Preferred lndicator range (grams) Solvent 1 {Brom Thymol Blue About 0.2 }About 25 ml. ethyl alcohol, (1.5. to about 100 Phenol Red, water-soluble About 0.2 ml. with distilled Water. 2.--; Neutral Red r About 0.45 About 32.5 ml. ethanol, q.s. to about 100 ml.

- with distilled Watre. Phenol Red, Water-soluble About 0.055.-. About 60 ml. ethanol, q.s. to about 100 ml. with distilled Water. 4. Rosolie acid About 0.4 About ml. ethanol, q.s. to about ml. with distilled water. 5 m-Dinitrobenzoylene urea About 0.5.-. Q.s. to about 100 ml. with distilled Water. 6 BrilliantYellow About 125.. Do

7 Quinoline Blue About 0.7"... About 75 ml. ethanol, q.s. to about 100 with a distilled water.

indicator or reagent zone by contact with fingers. If forceps are used or if only the barrier zone is handled, the three-zone variation of the product can be used.

For ease of operation, the preferred diagnostic product is prepared from a bibulous material which is divided into at least four zones. Zones 2 and 4 are impregnated with the hydrophobic barrier composition and allowed to dry. The reagent medium is applied to zone 1 and the indicator system is applied to zone 3. Both are allowed to dry. Care must be taken to see that the various zones do not overlap.

To facilitate packaging, the impregnated bibulous material is normally cut into narrow strips suitable for insertion into a test tube, each strip containing all the impregnated zones.

The test is performed by suspending a heavy loopful of organisms (3.0 mm. loop) from an agar media such as a Triple Sugar Iron Agar, Tryptic Soy Agar, or Nutrient Agar Slant in 0.3 ml. saline in a Kahn tube. The strip of bibulous material is inserted into the suspension of organisms in such a manner that zone 1, impregnated with the reagent medium, is in contact with the suspension of organisms, while zone 3, impregnated with the indicator zone, is not. The test tube containing the strip is incubated at 37 C. for four hours. The tube is then tilted to wet zone 3 of the strip with the suspension of organisms and bring the test system into contact with the indicator. The test tube is returned 'to normal position and held at room temperature for about 30 minutes.

When the diagnostic product contains the most preferred indicator combination (Brom Thymol Blue and Phenol Red), utilization of citrate or a positive reaction is indicated by a purple coloration of a shade corresponding to the Lewis Roberts Matchmaker Color Chart numbers MM272 (strong), MM294,. MM314, and MM315 (weak). A negative reaction is indicated by a brown color of a shade corresponding to the Lewis Roberts Matchmaker Color Chart numbers MM374 and MM353. (Lewis Roberts, Inc., 72 Union St., Newark, NJ.) Thus, a definite color change is obtained and the ambiguous bluegreen color which sometimes occurred when the previously used Hargrove-Weaver modification was applied to a paper strip is avoided. Moreover, the combined indicator solution gives a positive reaction at a lower pH (7.5) than the positive reaction (pH 7.6) of the Hargrove- Weaver test. This difference of 0.1 in pH is significant in this test since the alkaline metabolites produced by organisms utilizing citrate cause relatively small changes in pH. As a result, organisms which utilize citrate slowly show a more strongly positive reaction.

While the diagnostic test of the instant invention may be used to distinguish any organisms which typically utilize citrate from those which typically, do not utilize citrate, it is particularly elfective with certain genera and species in the Enterobacteriaceae.

In a clinical microbiology laboratory, the principal use for the citrate utilization test is in the differentiation of E. coli from Aerobacter-Klebsiella, and in the differentiation of Salmonella from Shigella.

The following examples are included in order to further illustrate this invention.

EXAMPLE 1 (A) Preparation of reagent medium Dissolve grams of sodium citrate (granules), 2.5

grams of monobasic potassium phosphate, and 0.5 gram 3 of dibasic potassium phosphate in distilled water to a. volume of ml. The pH is 6.1, and it is adjusted to pH 6.4 by adding 1 N sodium hydroxide. i r

(B) Preparation of indicator system meter). Adjust to a measuredpH of 6.4 by adding 0.1 N sodium hydroxide. Dissolve 0.2 gram Phenol ,Red, water soluble) Matheson Coleman and Bell) in. 5 0 milli liters of distilled water and mix with thepreviously prejpared solution of Brom Thymol Blue. The pH of the mixture is 7.7. Adjust to'a pH of 6.4 with 0.1 N hydrochloric acid.

(C) Preparation of the barrier composition? 85.0 milliliters Krylon' Crystal Clearis'diluted with 15.0 ml. of ethyl alcohol U.S.P. and mixed' thorortghly.

(D) Application to bibulous material" 'I;

Band 114 mm. (reagent zone) Band 2--l2.7 mm. (barrier zone) Band 3l4 mm. (indicator zone) Band 412.7 mm. (barrier zone) Band 529 mm. (blank zone) The Krylon barrier-composition of-Part Cin Examplel above is applied to Band 2' and to Band 4 in an amount sufiicient to saturate the paper in each bandSThe-sheet is allowed to dry in air to remove the solve'n from-the impregnated barrier bands.

The sheet is marked olf in 6.3 mm. sections perpendicular to the reference edge. Twenty-five sections (to be cut into the diagnostic test strips after impregnations' are completed) are obtained, eachtcontaining all five bands. The reagent medium from Part A in Example labove is placed in a Syringe Microbiuret.No.'Sl/4, LT,*calibrated to 0.200 microliter per division (Model-'-No. SB2,.Micro@ Meter Instrument Company, Cleveland, Ohio The dial is set to 45, which will emit 9.00 microliters. This amount is applied to each of the 25 sections previously marked off in Band 1 perpendicularly to the reference edge; .225 ml, is required. The sheet is allowed to dry ina The indicator medium from Part B 'in'Example-l above is placed in the above described microbiuret, set at 45, to give 9.00 microliters. This amountisapplied toeach of the 25 sections previously marked olf in Band"3," per p'en: dicularly to the reference edge. .225 jmliis re edQTheL sheet is allowed to dry in air. i The sheet is then cntinto the 6.3 mrnstrips' previously marked off, perpendicular to'the referenceedgel ii The diagnostic test strips are packed in testtubescontaining a desiccant. 1

E A P i Use of the diagnosticpaper strips .1 above,. is inserted in each.suspension.in such arnanner 'that the end of the 'strip containing the reagent; medium zone is in contact with the culture he tubeslare incu.

' bated at 37 C. for 4 hours after which the tuhe is tilted to wet the indicator zone. The tube is' kept at roomtemperature for 30 minutes and then read. A positive reaction is indicated by darkrto deep purplencolor Lewis Roberts Matchmaker .ECOlOIf Chart number; MM27 2 ,*MM294,1 MM314, and MM3 15. A negative reaction is indicated by moderate to strong brown (Lewis 'Robert' Matchmaker Color Chart number MM374 and MM353).

Results are listed below, along with reactions to the Simmons Citrate Test:

(3) from about 0.025 to abot 0.075 gram of Phenol Red (water soluble), from about to90 "Two of these cultures gave Weakly positive reactions.

The same strain or strains was'negative by both conventional and strip test. 3 One of these cultures gave a weakly positive reaction and gave a delayed positive reaction in the Simmons test.

The data obtained from the diagnostic strip test of this investigation is confirmed by the Simmons Citrate test for all organisms, except P. morganii, where the reactions of 11 species were atypical. There is no apparent explanationfor this deviation, but the results do not significantly afiect the utility of the diagnostic strip test, inasmuch as P. morganii would be shown as atypical.

It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of our invention.

Having described our invention, what We desire to secure by Letters Patent is:

1. A diagnostic preparation for the rapid and positive detection of citrate utilization by microorganisms which comprises a bibulous material having at least three zones, wherein there has been impregnated:

(A) In zone 1, 9 microliters of a reagent consisting essentially of:

(1) from about 8 to 12 grams of a source of citrate selected from the group consisting of citric acid, sodium citrate, and potassium citrate;

(2) a microbiologically inert butter selected from the group consisting of:

(a) about 2.5 grams of monobasic potassium phosphate and about 0.5 gram of dibasic potassium phosphate,

(b) about 4.23 grams of tris-(hydroxy methyl)aminomethane and about 4.16 grams of maleic acid, and

(c) about 1.1 grams of sodium borate and about 2.6 grams of monobasic potassium phosphate; and

(3) a sufiicient amount of distilled water to bring the volume of the solution to abut 100 ml.; said reagent system having a pH of about 6.4;

(B) In zone 2, a sufficient amount of a micro-biologicab ly inert hydrophobic barrier composition to saturate the bibulous material; and

(C) In zone 3, 9 microliters of at least one indicator system selected from the group consisting of:

(1) from about 0.18 to about 0.22 gram of Brom Thymol Blue, from about 0.18 to about 0.22 gram of Phenol Red (water soluble), from about 22.5 to about 27.5 ml. of a 1 to 4 carbon alcohol, and a sufiicient amount of distilled water to bring the volume of the solution to about 100 ml;

(2) from about 0.2 to about 0.7 gram of Neutral Red, from about 5 to about 60 ml. of a 1 to 4 carbon alcohol, and a suflicient amount of distilled water to bring the volume of the solution to about 100 ml.;

ml. of a 1 to 4 carbon alcohol, and a sufiicient amount of distilled water to bring the volume of the solution to about 100 ml.;

(4) from about 0.2 to about 0.6 gram of rosolic acid, from about to about ml. of a l to 4 carbon alcohol and a sufficient amount of distilled water to bring the volume of the solution to about 100 ml.;

(5 from about 0.3 to about 0.7 gram of m-dinitrobenzoylene urea, and a sufficient amount of distilled water to bring the volume of the solution to about 100 ml.;

(6) from about 0.75 to about 1.5 grams of Brilliant Yellow, and a sufficient amount of distilled water to bring the volume of the solution to about 100 ml.; and

(7) from about 0.2 to about 1.2 grams of Quinoline Blue, and a sufiicient amount of 50 to 100% ethyl alcohol to bring the volume of the solution to about 100 ml.;

said indicator system having a measured pH of about 6.4;

and wherein reagent system zone 1 is separated from indicator system zone 3 hydrophobic barrier zone 2.

2. A diagnostic preparation according to claim 1 having an additional barrier zone 4, contiguous only to indicator system zone 3, wherein there has been impregnated a sufficient amount of a microbiologically inert hydrophobic barrier composition to saturate the bibulous material.

3. A diagnostic preparation according to claim 1 wherein said reagent system consists essentially of:

(1) from about 8 to about 12 grams of sodium citrate,

(2) about 2.5 grams of monobasic potassium phosphate,

(3) about 0.5 grams of dibasic potassium phosphate,

and (4) a suflicient amount of distilled water to bring the volume of the solution to about 100 ml.; and said indicator system consists essentially of:

(1) from about 0.18 to about 0.22 gram of Brom Thymol Blue,

(2) from about 0.18 to about 0.22 gram of Phenol Red water soluble,

(3) from about 22.5 to about 27.5 ml. of an alcohol solvent selected from methyl, ethyl, propyl and butyl alcohol, "and (4) a sufficient amount of distilled water to bring the volume of the solution to about 100 ml.

4. A diagnostic preparation according to claim 3 wherein said reagent system consists essentially of (1) about 10 grams of sodium citrate,

(2) about 2.5 grams of monobasic potassium phosphate,

(3) about 0.5 gram of dibasic potassium phosphate and 1 1 (4) a sufiicient amount of distilled water to bring the volume of the solution to about 100 ml.; said hydrophobic barrier composition comprises:

(1) from about 75 ml. to about 100 ml of an acrylic resin coating composition, and

(2) from about to about 25 ml. of a lower alcohol diluent for said acrylic resin coating composition; and

said indicator system consists essentially of:

(1) about 0.2 gram of Brom Thymol Blue,

(2) about 0.2 gram of Phenol Red water soluble,

(3) about 25 m1. of ethyl alcohol, and

(4) a sufficient amount of distilled water to bring the volume of the solution to about 100 ml.

5. A process for the detection of citrate utilization by microorganisms comprising:

(A) allowing zone 1 of the diagnostic preparation of claim 1 to come into contact with a saline suspension of an unknown culture for from about 3 hours to about 6 hours, at a temperature ranging from about 34 C. to about 37 C.;

(B) allowing the above-treated suspension to come into contact with zone 3 of the aforementioned diagnostic preparation; and

(C) allowing at least minutes to elapse for a color to develop as an indication of positive or negative citrate utilization by'the unknown culture? 6. A process according to claimSf wherein the unknown culture is brought into contact with-zone 1 of the diagnos: tic preparation for about'4 hours at-a'temperature-of about 37 C. i

References Cited UNITED, STATES PATENTS 9/ 1967 Evans et al OTHER REFERENCES Hargrove et al., Am, ,J. ClinLPathi' 21,:

LIONEL M. sHAP u rgiina y M. D. HENSLEY, Assistant Examiner US. Cl. X.R. 

